CN103424546B - Specific identification method for biological active components in deer leather gel - Google Patents
Specific identification method for biological active components in deer leather gel Download PDFInfo
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- CN103424546B CN103424546B CN201210154456.4A CN201210154456A CN103424546B CN 103424546 B CN103424546 B CN 103424546B CN 201210154456 A CN201210154456 A CN 201210154456A CN 103424546 B CN103424546 B CN 103424546B
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Abstract
The invention discloses a specific identification method for biological active components in deer leather gel. The identification comprises an enzyme linked immunosorbent assay (ELISA) for specific deer collagen in the deer leather gel, wherein the ELISA is used for determining whether the deer leather gel contains the specific deer collagen according to the color result and detecting the content of the specific deer collagen; adopts a ninhydrin post column derivation method, which is used for carrying out quantitative and qualitative analysis identifications for specific amino acid composition in deer leather gel; and carries out a PCR amplification test identification for deer leather gel specific DNA sequence in deer leather gel, and if stripes which represent the deer specific protein components can be amplified out, indicating that the sample to be test is a qualified product. The method mentioned above provides a scientific and reliable identification method for distinguishing true or false and high-quality or low-quality of deer leather gel products in the market, and detecting the content of effective biological components in deer leather gel.
Description
Technical field
The present invention relates to a kind of authentication method of pure deerskin glue bioactive ingredients, particularly relate to the specificity identification method of special collagen, Specific amino acid and specific DNA contained in a kind of deerskin glue.
Background technology
A large amount of record is had to the qi-restoratives damage of deer goods, benefiting essence-blood, strong effect such as waist kidney and tranquilizing mind in Chinese traditional medicine.Then to the science data of deerskin glue, particularly to the bioactive ingredients contained by deerskin glue, specificity identification as collagen, amino acid and DNA still has no data available, adopt the bioactive ingredients of high-tech to deerskin glue to measure, contribute to the quality producing and differentiate deerskin glue product.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, to the invention provides in a kind of deerskin glue the specificity identification method of contained bioactive ingredients, the method is that the true and false quality of deerskin glue product on Vehicles Collected from Market provides science and identifies mode reliably.
The present invention in order to the technical scheme solving its technical matters and adopt is:
A specificity identification method for contained bioactive ingredients in deerskin glue, comprises the enzyme linked immunosorbent assay to special deer collagen albumen contained in deerskin glue; Adopt triketohydrindene hydrate post-column derivation method to Specific amino acid composition analysis qualification contained in deerskin glue; With the pcr amplification experimental identification to deerskin glue specific DNA sequence contained in deerskin glue.
The described enzyme linked immunosorbent assay to special deer collagen albumen contained in deerskin glue, uses the antibody immunoglobulin of special deer collagen albumen as coated antibody, adopts peroxidase labelling special deer collagen protein antibodies immunoglobulin (Ig).
The described enzyme linked immunosorbent assay to special deer collagen albumen contained in deerskin glue, comprises the following steps:
With the antibody immunoglobulin bag of special deer collagen albumen by polypropylene reaction plate, and after bag is done, wash away the antibody immunoglobulin coating buffer of described special deer collagen albumen; Testing sample and the antibody immunoglobulin of the special deer collagen albumen be coated on polypropylene reaction plate are carried out antigen-antibody reaction, and wash away described testing sample after above-mentioned antigen-antibody reaction terminates; Special deer collagen protein antibodies immunoglobulin (Ig) described in peroxidase labelling is adopted to form the special deer collagen protein antibodies immunoglobulin (Ig) of peroxidase labelling; The special deer collagen protein antibodies immunoglobulin (Ig) of above-mentioned oxide enzyme labeling is added on above-mentioned polypropylene reaction plate, forms the compound of the special deer collagen protein antibodies immunoglobulin (Ig) of special deer collagen protein antibodies immunoglobulin (Ig)-special deer collagen albumen-peroxidase labelling; O-phenylene diamine substrate is adopted to develop the color; If develop the color successfully, show containing special deer collagen albumen in testing sample, adopt enzyme mark color comparator to measure the content of special deer collagen albumen in the quantitative sample of light absorption value of colour developing thing.
The described pcr amplification experimental identification to deerskin glue specific DNA sequence contained in deerskin glue, comprises the following steps:
By traditional DNA purification method, the genomic DNA in deerskin glue is carried out purification, obtain purified deerskin glue extracting genome DNA thing; Utilize primer pair CAGCTAGCTATAGTCT/GTATGGATAATGCAGTT to carry out pcr amplification test to gained deerskin glue extracting genome DNA thing to increase, obtain amplified production one; Utilize primer pair CGTGGACTACGATGCTT/TAGTCCTTGGCTACGT to carry out pcr amplification test to gained deerskin glue extracting genome DNA thing to increase, obtain amplified production two; Gained amplified production one and two is carried out agarose gel electrophoresis detection respectively, judges the true and false of deerskin glue according to amplified production clip size.
Advantageous Effects of the present invention is: the present invention is by the enzyme-linked immunosorbent assay to special deer collagen albumen contained in deerskin glue, judge whether containing described special deer collagen albumen in deerskin glue according to colour developing result, and the content of contained special deer collagen albumen; Triketohydrindene hydrate post-column derivation method is adopted to carry out quantification and qualification qualification to Specific amino acid composition contained in deerskin glue; Pcr amplification experimental identification with to deerskin glue specific DNA sequence contained in deerskin glue, if can amplify the band representing deer differential protein composition, shows that testing sample is certified products; Said method is that the true and false of deerskin glue product on Vehicles Collected from Market is good and bad, and in deerskin glue, contained effectively biotic component content provides the reliable authentication method of science.
Accompanying drawing explanation
The protein concentration typical curve of Fig. 1 for formulating when adopting enzyme mark color comparator to measure special deer collagen albumen in enzyme-linked immunosorbent assay;
Fig. 2 is amino acid contained in pure deerskin glue (except proline) HPLC analysis chart;
Fig. 3 is contained proline HPLC analysis chart in pure deerskin glue;
Fig. 4 is the agarose gel electrophoresis result of contained genomic DNA amplification product in pure deerskin glue, and the primer pair that wherein A uses is DF/DR(and EP-2/H1478), the primer pair that B uses is SF/SR(and EP-1/H1478).
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
(1) enzyme-linked immunosorbent assay (ELISA) measures special deer collagen albumen contained in deerskin glue, wherein adopted testing sample lysate is respectively the deerskin glue solution of 50mg/ml and the deerskin glue dry powder solution of 50mg/ml, in 96 hole polypropylene reaction plates, adopt two secondary orifices, the method comprises the following steps:
(1) by antibody immunoglobulin (the monoclonal anti-Collagen Type 1 of available from Sigma of special deer collagen albumen, production number is C2456) wrap by 96 hole polypropylene reaction plates, at bag is placed in 4 DEG C by good reaction plate after 24h, automatic plate washer (U.S. Biorad-680 model) is adopted to wash away the antibody immunoglobulin coating buffer of special deer collagen albumen;
(2) by each testing sample lysate respectively after diluting 20 times and 50 times, add in two subsidiary reaction holes of the polypropylene reaction plate after (1) process respectively, hatch 1h at 37 DEG C after, the dilution of each testing sample lysate is discarded, and adopt automatic plate washer washing reaction plate;
(3) in two secondary orifices of the polypropylene reaction plate after (2) process, the special deer collagen protein antibodies immunoglobulin (Ig) marked through horseradish peroxidase (HRP) is added, again carry out hatching 1h at 37 DEG C, hatch after terminating and adopt automatic plate washer washing reaction plate;
(4) in two secondary orifices of the polypropylene reaction plate after (3) process, o-phenylenediamine (OPD) substrate solution is added into, adopt enzyme mark color comparator (the Spectra Max M5 that Molecular Devices company produces) to measure after wait colour developing, containing special deer collagen albumen in every gram of pure deerskin glue of the present invention is 350mg.
The Cleaning Principle of above-mentioned steps is as described below: on polypropylene reaction plate, bag is by special deer collagen protein antibodies immunoglobulin (Ig), after adding testing sample lysate again in the reacting hole of reaction plate, if containing special deer collagen albumen in testing sample lysate, then special deer collagen albumen can form the compound one of special deer collagen albumen-special deer collagen protein antibodies immunoglobulin (Ig) with special deer collagen protein antibodies immunoglobulin (Ig), when then adding the special deer collagen protein antibodies immunoglobulin (Ig) through HRP peroxidase labelling in above-mentioned compound, the compound two of the special deer collagen protein antibodies immunoglobulin (Ig) of special deer collagen protein antibodies immunoglobulin (Ig)-special deer collagen albumen-HRP peroxidase labelling can be formed.After add OPD substrate solution in above-mentioned compound two system, color reaction can be there is in OPD with peroxidase, according to the shade that enzyme mark color comparator detects, compared with the typical curve formulated on enzyme mark color comparator, draw the content of contained special deer collagen albumen in deerskin glue.
As shown in Figure 1, and to carry out contrasting protein content in rear gained sample as shown in table 1 with this typical curve for the protein concentration typical curve formulated when adopting in above-mentioned steps (4) enzyme mark color comparator to measure:
In table 1 deerskin glue, contained special collagen concentration measures
(2) triketohydrindene hydrate post-column derivation method measures amino acid contained in deerskin glue, comprises the following steps:
(1), after adopting ultrapure water to be diluted to 1mg/ml the deerskin glue sample dissolution liquid to be measured of 50mg/ml, miillpore filter is used to filter described dilution, using gained filtered fluid as need testing solution;
(2) by gained need testing solution microsyringe in (1) to sample introduction 20.0 μ l in amino-acid analyzer (the full-automatic amino-acid analyzer of L-8800 type that HIT produces), the chromatographiccondition of described amino-acid analyzer is as described below: chromatographic column adopts the cation exchange column of 4.6mm × 60mm, model is Hitachi 2622SC, mobile phase selects citrate three sodium damping fluid, quaternary gradient eluent pH value is 3.2 ~ 4.9, determined wavelength is 570nm, and eluent flow rate is 0.4ml/min; Column temperature is 50 DEG C; The condition of post-column derivation is: reactant liquor A:L-propylene glycol monomethyl ether, 390g indenes diketone; Reactant liquor B propylene glycol monomethyl ether-sodium acetate, acetic acid (40:60, V/V); Temperature of reaction is 135 DEG C, and reactant liquor flow velocity is 0.35ml/min, and the scale factor adopted is 1:1000 and 1:2000.
The principle that above-mentioned automatic amino acid analyzer method carries out amino acid whose component analysis is as described below: utilize various amino acid whose Acidity of Aikalinity, polarity and molecular size range not congruence property, Zeo-karb is used to be separated on a column, after sample liquid adds chromatographic column top, adopt the buffer solution of different pH value and ion concentration they can be eluted successively.Acidic amino acid and the larger amino acid of polarity before this, being secondly nonpolar aromatic amino acid, is finally basic amino acid; Large being first eluted of ratio molal weight that molal weight is little, the amino acid eluted can develop the color with triketohydrindene hydrate, thus quantitative each seed amino acid.The amino acid whose foundation of quantitative measurement is that amino acid and ninhydrin reaction generate bluish violet compound, and the shade of this compound is directly proportional with each corresponding amino acid whose content, and wherein proline and hydroxyproline then generate yellowish-brown compound with triketohydrindene hydrate.According to the shade that color comparator detects, compared with the amino acid typical curve formulated on color comparator, draw content amino acid contained in testing sample.In deerskin glue, the stratographic analysis figure of amino acid classes is shown in shown in Fig. 2 and Fig. 3, and wherein Fig. 3 is contained proline HPLC analysis chart in deerskin glue; Amino acid analysis result is as shown in table 2, and the computing method of wherein said amino acid content are the mark that each amino acid content accounts for respective substance total amount, wherein
※represent that this seed amino acid is essential amino acid.
Amino acid content contained in table 2 deerskin glue
| Amino acid classes | Amino acid content (%) |
| Asp(L-aminobutanedioic acid) | 6.42 |
| Thr ※(threonine) | 2.21 |
| Ser(serine) | 3.38 |
| Glu(glutamic acid) | 11.17 |
| Pro(proline) | 13.81 |
| Gly(glycocoll) | 23.80 |
| Ala(alanine) | 9.26 |
| Cys(cystine) | 0.29 |
| Val ※(valine) | 2.64 |
| Met ※(methionine) | 0.59 |
| Ile ※(isoleucine) | 1.44 |
| Leu ※(leucine) | 3.51 |
| Tyr(tyrosine) | 1.04 |
| Phe ※(phenylalanine) | 2.02 |
| Lys ※(lysine) | 4.00 |
| His(histidine) | 0.77 |
| Arg(arginine) | 7.81 |
| NH3(ammonia) | (0.74) |
| Total amino acid content | 94.16 |
As shown in table 2, containing 17 kinds of different amino acid in pure deerskin glue, wherein threonine, valine, methionine, isoleucine, leucine, phenylalanine and lysine be the mankind can not the essential amino acid of self synthesis; In addition the content of glycocoll is 23.8%.
(3) the pcr amplification test for identification of specific DNA sequence in deerskin glue, comprises the following steps:
(1) DNA extraction kit adopting QLAGEN company to produce, carries out the extraction purification of contained genomic dna sequence in deerskin glue, obtains purified deerskin glue extracting genome DNA thing according to the operation instruction on described kit;
(2) (1) gained extracting genome DNA thing is carried out PCR reaction under following reaction system and reaction conditions: reaction system is extracting genome DNA thing 1 μ g; 10 × PCR Buffer is 10 μ l; Taq.DNA polymerase is 3U; Distilled water complements to 100 μ l; Primer pair is each 80pmol of forward and reverse sequence of often pair of primer pair.Wherein Mg
2+content is 1.5mmol/L; DNTPs mixture content is often kind of each 200 μm of ol/L; Wherein primer pair adopts EP-1/H1478(SF/SR) or primer pair EP-2/H1478(DF/DR), forward and reverse sequence of described SF/SR primer pair is: CGTGGACTACGAT GCTT; TAGTCCTTGGCTACGT;
Forward and reverse sequence of described DF/DF primer pair is: CAGCTAGCTATAGTCT; GTATGGATAATGCAGTT.Response procedures is 97 DEG C of sex change 7min; Again after the 30sec that anneals at 60 DEG C, 68 DEG C of downward-extension 50sec, PCR instrument (the MJ Mini model that Bio-Rad company produces) carries out 30 amplification cycles;
(3) pcr amplification product by (2) gained is carried out agarose gel electrophoresis detection: adopt 1wt.% Ago-Gel, produces with DL200 DNA Maker(Takara company) in DNA fragmentation be the size that molecular weight standard (wherein DNA fragmentation length be 100bp, 00 bp, 300 bp, 400 bp, 500 bp and 1000bp) calculates pcr amplification product fragment.The agarose gel electrophoresis result of amplified production as shown in Figure 4.
Sequence table
<110> Suzhou City HongGuanZhuang GuoYao herbal pieces prepared for decoction Co., Ltd
The specificity identification method of contained bioactive ingredients in <120> deerskin glue
<130> 2012
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> artificial sequence
<400> 1
cgtggactac gatgctt17
<210> 2
<211> 16
<212> DNA
<213> artificial sequence
<400> 2
tagtccttgg ctacgt16
<210> 3
<211> 16
<212> DNA
<213> artificial sequence
<400> 3
cagctagcta tagtct16
<210> 4
<211> 17
<212> DNA
<213> artificial sequence
<400> 4
gtatggataa tgcagtt17
Claims (2)
1. a specificity identification method for bioactive ingredients contained by deerskin glue, is characterized in that:
Comprise the enzyme linked immunosorbent assay to special deer collagen albumen contained in deerskin glue, wherein use the antibody immunoglobulin of special deer collagen albumen as coated antibody, adopt peroxidase labelling special deer collagen protein antibodies immunoglobulin (Ig); Adopt triketohydrindene hydrate post-column derivation method to Specific amino acid composition analysis qualification contained in deerskin glue; With the pcr amplification experimental identification to deerskin glue specific DNA sequence contained in deerskin glue;
The described enzyme linked immunosorbent assay to special deer collagen albumen contained in deerskin glue, comprise the following steps: with the antibody immunoglobulin bag of special deer collagen albumen by polypropylene reaction plate, and after bag is done, washes away the antibody immunoglobulin coating buffer of described special deer collagen albumen; Testing sample and the antibody immunoglobulin of the special deer collagen albumen be coated on polypropylene reaction plate are carried out antigen-antibody reaction, and wash away described testing sample after above-mentioned antigen-antibody reaction terminates; Special deer collagen protein antibodies immunoglobulin (Ig) described in peroxidase labelling is adopted to form the special deer collagen protein antibodies immunoglobulin (Ig) of peroxidase labelling; The special deer collagen protein antibodies immunoglobulin (Ig) of above-mentioned oxide enzyme labeling is added on above-mentioned polypropylene reaction plate, forms the compound of the special deer collagen protein antibodies immunoglobulin (Ig) of special deer collagen protein antibodies immunoglobulin (Ig)-special deer collagen albumen-peroxidase labelling; O-phenylene diamine substrate is adopted to develop the color; If develop the color successfully, show containing special deer collagen albumen in testing sample, adopt enzyme mark color comparator to measure the content of special deer collagen albumen in the quantitative sample of light absorption value of colour developing thing.
2. the specificity identification method of bioactive ingredients contained by deerskin glue according to claim 1, is characterized in that: the described pcr amplification experimental identification to deerskin glue specific DNA sequence contained in deerskin glue, comprises the following steps:
By traditional DNA purification method, the genomic DNA in deerskin glue is carried out purification, obtain purified deerskin glue extracting genome DNA thing; Utilize primer pair CAGCTAGCTATAGTCT/GTATGGATAATGCAGTT to carry out pcr amplification test to gained deerskin glue extracting genome DNA thing to increase, obtain amplified production one; Utilize primer pair CGTGGACTACGATGCTT/TAGTCCTTGGCTACGT to carry out pcr amplification test to gained deerskin glue extracting genome DNA thing to increase, obtain amplified production two; Gained amplified production one and two is carried out agarose gel electrophoresis detection respectively, judges the true and false of deerskin glue according to amplified production clip size.
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| CN104483486A (en) * | 2014-12-22 | 2015-04-01 | 扬州大学 | Kit for identifying deer antlerblood slide |
| CN105738632B (en) * | 2016-02-24 | 2017-06-23 | 浙江理工大学 | A kind of detection method for distinguishing sheepskin species |
| CN113004374B (en) * | 2018-09-03 | 2023-01-31 | 贵州广济堂健康药业有限公司 | Deer glue characteristic peptide and method for identifying deer glue contained in sample to be detected |
| CN111060622B (en) * | 2019-12-26 | 2021-06-29 | 北京化工大学 | Labeled peptide of tuna-derived component and application of labeled peptide in detection of collagen degradation product and product thereof |
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| WO2004021007A1 (en) * | 2002-08-29 | 2004-03-11 | Invitek Gesellschaft Für Biotechnologie & Biodesign Mbh | Elisa kits for detecting collagenase 3 as a proenzyme and in an activated form in body fluids and cell culture supernatants |
| CN101265500A (en) * | 2008-02-29 | 2008-09-17 | 华东理工大学 | PCR determination method for Chinese medicine or Chinese medicinal crops derived from eukaryote |
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| WO2004021007A1 (en) * | 2002-08-29 | 2004-03-11 | Invitek Gesellschaft Für Biotechnologie & Biodesign Mbh | Elisa kits for detecting collagenase 3 as a proenzyme and in an activated form in body fluids and cell culture supernatants |
| JP2009014695A (en) * | 2007-06-29 | 2009-01-22 | Morisuke Yokoyama | Type iv collagen nc1 measurement kit |
| CN101265500A (en) * | 2008-02-29 | 2008-09-17 | 华东理工大学 | PCR determination method for Chinese medicine or Chinese medicinal crops derived from eukaryote |
| JP2010271295A (en) * | 2009-05-24 | 2010-12-02 | Morisuke Yokoyama | Measuring reagent |
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Address after: Kunshan City Qiandeng Town Suzhou city of Jiangsu province from 215343 Ma Fumin Road No. 89 Applicant after: SUZHOU HONGGUANZHUANG MEDICINES CO., LTD. Address before: Kunshan City Qiandeng Town Suzhou city of Jiangsu province from 215343 Ma Fumin Road No. 89 Applicant before: Hongguangzhuang Chinese Herbal Piece Co., Ltd., Suzhou |
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Free format text: CORRECT: APPLICANT; FROM: HONGGUANGZHUANG CHINESE HERBAL PIECE CO., LTD., SUZHOU TO: SUZHOU HONGGUANZHUANG TRADITIONAL CHINESE MEDICINE CO., LTD. |
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